Cancer progression involves disordered gene regulation. In epithelial tissues such as skin, where ~90% of human malignancies arise, the earliest steps in this process involve disrupted expression of differentiation and proliferation genes, leading to the abnormal tissue polarity that presages cancerous invasion. The genome-wide nature of the gene dysregulation seen in the tumor progression process implicates globally active epigenetic gene regulators, however, each regulator class contains multiple isoforms whose specific roles in cancer are not fully understood. In the case of DNA methylation, there are multiple non-redundant DNA methyltransferases (DNMTs), which act primarily by silencing expression of specific genes. DNMT action is antagonized by a group of genes found over the past 2 years to actively promote DNA demethylation. The role of DNMTs and their antagonists in early tumor progression in human tissue, however, is unknown. This revised proposal focuses on characterizing the role of regulators of DNA methylation in early tumor progression, specifically the conversion of normal human epidermis to invasive neoplasia. First, we will define the role of DNMT isoforms in early tumor progression. We recently observed that DNMT1, which is mis-expressed in squamous cell carcinoma (SCC) of skin and other tissues, controls the normal epidermal growth and differentiation gene program and regulates a significant subset of the genes that are altered in early human epidermal tumorigenesis. Using an inducible human tissue model of Ras-driven epidermal neoplasia, in Aim I we will test an additive DNMT model in which DNMT1 maintains the undifferentiated proliferative state while DNMT3A/B/L silence de novo induction of tumor inhibitory genes. Second, we will study the role of recently characterized DNMT-antagonizing proteins. Active DNA demethylation has recently been found to proceed via proteins interacting with the base excision repair (BER) pathway (MBD4, TDG, and AID/Apobec2) and the nucleotide excision repair (NER) pathway (XPA, XPG, ERCC2, TAF12). Gadd45 proteins interact with both BER and NER mechanisms to enable DNA demethylation. In support of a potential role for mediators of DNA demethylation in tumorigenesis, we recently observed that Gadd45 promotes epidermal differentiation and inhibits ectopic tumor formation in association with diminishing DNA methylation at specific genes. In Aim II, we will test the hypothesis that specific mediators of DNA demethylation inhibit early tumor progression. At the end of proposed funding, we plan to have characterized the role of regulators of DNA methylation on tumor progression in human tissue as a foundation for future strategies for cancer prevention and treatment. 1 PUBLIC HEALTH RELEVANCE: Conversion of normal tissue into cancer involves progressively disordered gene expression. Epigenetic regulators possess the capacity to control global gene expression in cancer and other processes by altering genomic marks, such as DNA methylation. In epithelial tissues, such as skin, where ~90% of human malignancies arise, regulators of DNA methylation are abnormally expressed, however, their roles in cancer progression are undefined. The current proposal aims to characterize the role of regulators of DNA methylation on tumor progression in human tissue as a foundation for future strategies for cancer prevention and treatment. 1